The invention relates to mechanisms and methods used to form a microarray of multiple probes used to detect the presence of a target biological material or a target chemical.
A microarray is an array of spots of biological or chemical samples (xe2x80x9cprobesxe2x80x9d) immobilized at predefined positions on a substrate. Each spot contains a number of molecules of a single biological or chemical material. To interrogate the array, the microarray is flooded with a fluid containing one or more biological or chemical samples (the xe2x80x9ctargetxe2x80x9d), elements of which typically interact with one or more complementary probes on the microarray. In DNA microarrays in particular, the probes are oligonucleotide or cDNA strains, and the target is a fluorescent or radioactive-labeled DNA sample. The molecular strands in the target hybridize with complementary strands in the probe microarray. The hybridized microarray is inspected by a microarray reader, which detects the presence of the radioactive labels or which stimulates the fluorescent labels to emit light through excitation with a laser or other energy sources. The reader detects the position and strength of the label emission in the microarray. Since the probes are placed in predetermined and thus known positions in the microarray, the presence and quantity of target sequences in the fluid are identified by the position at which fluorescence or radiation is detected and the strength of the fluorescence or radiation.
Microarray technology provides an extremely useful tool to conduct biological or chemical experiments in a massive parallel fashion because of the large number of different probes that one can fabricate onto the microarray. It is particularly powerful in screening, profiling and identifying DNA samples.
Microarrays today come as two-dimensional probe matrices fabricated on solid glass or nylon substrates. Because the target samples are generally hard to produce or very expensive, it is highly desirable to perform assays on as many features as possible on a single microarray. This calls for a significant increase in probe density and quantity on a single substrate. In general, microarrays with probe pitch smaller than 500 xcexcm (i.e. density larger than 400 probes per sqr. centimeter) is referred as high density microarrays, otherwise, they are xe2x80x9clow densityxe2x80x9d microarrays.
There are two microarray fabrication techniques on the market, photolithographic and robotic spotting techniques. The photolithographic technique [U.S. Pat. Nos. 5,445,934, 5,744,305] adapts the same fabrication process for electronic integrated circuits to synthesize probes in situ base by base. This technique requires a large capital outlay for equipment running up to hundreds of millions of dollars. The initial setup of new microarray designs is also very expensive due to the high cost of producing photo masks. This technique is therefore only viable in mass production of standard microarrays at a very high volume. Even at high volumes, the complexity in synthesis still limits the production throughput resulting in a high microarray cost. This complexity also limits the length of the synthesized DNA strain to the level of a short oligonucleotide (xcx9c25 bases), which reduces the specificity and sensitivity of hybridization in some applications.
The established robotic spotting technique [U.S. Pat. No. 5,807,522] uses a specially designed mechanical robot, which produces a probe spot on the microarray by dipping a pin head into a fluid containing an off-line synthesized DNA and then spotting it onto the slide at a predetermined position. Washing and drying of the pins are required prior to the spotting of a different probe in the microarray. In current designs of such robotic systems, the spotting pin, and/or the stage carrying the microarray substrates move along the XYZ axes in coordination to deposit samples at controlled positions of the substrates. Because a microarray contains a very large number of different probes, this technique, although highly flexible, is inherently very slow. Even though the speed can be enhanced by employing multiple pin-heads and spotting multiple slides before washing, production throughput remains very low. This technique is therefore not suitable for high volume mass production of microarrays.
In addition to the established quill-pin spotting technologies, there are a number of microarray fabrication techniques that are being developed. These include the inkjet technology and capillary spotting.
Inkjet technology is being deployed to deposit either cDNA/oligonucleotides, or individual nucleotides at defined positions on a substrate to produce an oligonucleotide microarray through in situ synthesis. Consequently, an oligonucleotide is produced in situ one base at a time by delivering monomer-containing solutions onto selected locations, reacting the monomer, rinsing the substrate to remove excess monomers, and drying the substrate to prepare it for the next spot of monomer reactant.
An emerging spotting technique uses capillaries instead of pins to spot DNA probes onto the support. Four references discuss capillary-based spotting techniques for array fabrication:
WO 98/29736, xe2x80x9cMultiplexed molecular analysis apparatus and methodxe2x80x9d, by Genometrix Inc.
WO 00/01859, xe2x80x9cGene pen devices for array printingxe2x80x9d, by Orchid Biocomputer Inc.
WO 00/13796, xe2x80x9cCapillary printing systemxe2x80x9d, by Incyte Pharmaceuticals Inc.
WO 99/55461, xe2x80x9cRedrawn capillary imaging reservoirxe2x80x9d, by Corning Inc.
In summary, due to the high cost of production, microarrays fabricated with existing technologies are far too expensive as a single use lab supply.
The invention provides a probe printing system having a print head composed of one or more bundles of randomly bundled or discretely bundled capillaries as described herein. A bundle of capillaries has a portion where at least the proximal ends of the capillaries are immobilized in a planar matrix and a facet is formed for printing. The immobilized portion is preferably sufficiently rigid that it may be used to print a probe microarray upon a substrate with minimal or no deformation (deformation may result in portions of the microarray not being printed to the substrate). The immobilized portion is therefore sufficiently rigid to ensure good contact with the substrate across the portion of the facet in contact with the substrate. The distal ends of the capillaries may be free or may be attached to reservoirs. The capillaries include, but are not limited to, fiber optic or other light-conducting capillaries, through which light as well as fluid can be conveyed; and other flexible or rigid capillaries.
A capillary bundle in one embodiment of the invention has a plurality of individual capillaries having proximal and distal ends. The outer diameter of a capillary is typically less than about 300 micron, preferably the outer diameter is less than about 100 micron. Each of the capillaries of the bundle has a channel extending from the proximal end to the distal end of the capillary, and each of the capillaries has a channel-facing wall. The channel diameter is preferably less than 100 micron.
A bundle of individual capillaries is distinguished from a unitary structure in which tubular preforms are fused to one another to form a large array of preforms and then stretched to form a unitary array of channels.
The proximal ends of capillaries of a bundle may be secured to one another in a solid mass such that the proximal ends of the capillaries are substantially coplanar at a facet of the solid mass. Proximal ends are substantially coplanar when liquid flowing through the capillaries form spots on a flat surface of the substrate when the facet of the solid mass is either pressed against the surface or is in sufficient proximity to the surface that droplets from the capillaries are deposited on the surface. Generally, proximal ends are substantially coplanar when all ends terminate within about 100 microns of one another. Preferably, proximal ends terminate within about 50 microns of one another. More preferably, proximal ends terminate within about 20 microns of one another. Even more preferably, proximal ends terminate within 5 microns of one another.
A capillary bundle may contain any number of capillaries. Preferably, the bundle contains at least about 1000, 5000, 10,000, 50,000, 100,000, or 500,000 capillaries. A capillary bundle also preferably contains at least about 83, 416, 500, 833, 1000, 4166, 5,000, 8333, 41,666, 10,000, 20,000, or 40,000 capillaries per cm2 that print non-overlapping spots on a substrate.
Capillaries of the bundle may individually have a well formed at their distal ends. Such wells may be formed by etching the proximal end of a silica capillary that has a region near the channel of the capillary that is doped compared to the region nearer the outer wall. The facet of the solid mass may be coated with an electrically-conductive material to facilitate establishing a potential difference that moves probe molecules. Each of the capillaries may have a substantially uniform inner diameter from their distal ends to their proximal ends, and each of the capillaries preferably has substantially the same diameter. This assures a uniform flow rate of fluid through the capillaries, so that spot sizes are approximately equal and so that individual spots do not join together and mix. Preferably, the diameter along a capillary has no more than about 10%, more preferably no more than about 3% variation, and preferably the diameters of all of the capillaries are within about 10%, more preferably about 3% of the mean diameter of the capillaries.
The invention also provides methods of making capillary bundles, methods of correlating the myriad number of individual capillaries of a print head to the reservoirs to which they are attached, and methods of printing microarrays using any of the printing systems, capillaries, and print heads further described herein.
A capillary bundle may be formed by a number of different methods. In one method, individual capillaries are gathered together in no particular order and secured to one another to form a random bundle. In such a random bundle, the distal ends of the capillaries are grouped in a first arrangement, the proximal ends of the capillaries are grouped in a second arrangement, and the first arrangement is not identical to the second arrangement. Often, it is not possible to know which distal end corresponds to which proximal end in such a random bundle until the proximal ends and the distal ends are registered to one another.
The proximal and distal ends of the capillaries may be registered to one another using any of a number of methods. If the capillaries are light-conducting capillaries, light may be launched into a distal end of each capillary and the position of light exiting the proximal end of the capillary is noted and recorded. Other methods include registering the position using a temperature change induced by an air or another fluid flowing through the capillary or by visually observing e.g. an ink that passes through the capillary.
In another method, individual capillaries are secured to one another to form an ordered bundle. In an ordered bundle, the correlation between distal ends and proximal ends is known at the time the ordered bundle is made. No registration of distal and proximal ends is necessary. In one method of making an ordered bundle, individual capillaries are inserted into a guide plate or a set of guide plates, and the capillaries at or near the proximal and/or distal ends or over most or all of the capillaries"" lengths are bonded together in a solid mass using, e.g., epoxy. The ends or capillaries may optionally be fused to form the solid mass. The guide plate or plates may be removed, since a sufficient portion of the capillaries are bonded or fused together in a solid mass at the point that the guide plates are removed. Removal of the guide plate forms a facet of the solid mass.
A print head of the invention has a capillary bundle as described herein attached or secured to a frame that is adapted to hold the capillary bundle in a print system. A print head may alternatively have a frame that holds a plurality of capillary bundles.
A print system has a print head and a plurality of reservoirs (such as those contained in a microtiter plate) in fluid communication with distal ends of the capillary bundle of the print head. A print system may have a voltage source connected to an electrically-conductive material on a facet of the print head and to an electrically conductive material contacting the probe-containing liquid near the proximal ends of the capillaries. A voltage regulator may be used to regulate the voltage and thus the rate of deposition of probe molecules.
Another print system of the invention may have a print head, a plurality of reservoirs, and a magnetic field generator that is positioned sufficiently closely to the print head to move a magnetic probe-containing fluid (such as a fluid containing magnetic beads or paramagnetic beads having probes attached to their surfaces) through the capillaries of the bundle.
A print system may have a flexible mount on which the substrate, the print head, or both are mounted. A flexible mount permits the substrate and/or print head to move and align themselves to one another to provide for improved print quality.
The print head of a print system may be configured so that it moves in only one direction (toward and away from the substrate on which probes are to be printed, or in the z-direction of an x-y-z coordinate system), with the substrates moving beneath the print head. Alternatively, the print head may be configured to move in all directions or to be stationary, with substrates being moved to the print head.
The reservoirs of a print system of the invention preferably reside in fixed positions, whereas the print head of the print system is free to move. Consequently, the capillaries of the capillary bundle of the print system have sufficient flexibility to allow capillary movement without requiring the reservoirs to also move. In addition, the reservoirs of a print system of the invention preferably reside in a regulated pressure chamber, wherein change of pressure moves solution in or out of the capillaries.
The invention provides a probe microarray comprising an arrangement of non-identical probes on a substrate in a honeycomb pattern, wherein, at the same center-to-center pitch, the density of probes is higher than that in a chessboard pattern. By xe2x80x9choneycombxe2x80x9d is meant a pattern of regular triangles and regular hexagons wherein each spot is at the center of a regular hexagon formed by six neighboring spots of equal distance to the center. The substrate may be porous or nonporous.
The invention further provides a probe microarray comprising a random arrangement of non-identical probes on a substrate. A random arrangement of non-identical probes is one in which probes on a substrate may appear to be organized locally into columns and rows or in a honeycomb pattern, but the probes do not have column and row order or honeycomb pattern across the entire microarray as is found in an array that is fabricated on a substrate using photolithographic techniques or robotic spotting techniques. Further, the individual probes of a first probe microarray having a random arrangement of non-identical probes printed using a first random bundle of capillaries will have positions on the substrate that differ from the positions of the same individual probes of a second probe microarray printed using a second random bundle of capillaries. The spatial positions of the individual probes are determined by the order and spatial relationship of the individual capillaries of the random bundle, and the order and spatial relationship of the individual capillaries in the bundle are random. A probe microarray printed using a random bundle is one example of a probe microarray made by placing non-identical probes on a substrate in a random pattern.
The probes are printed on print surface of the substrate, and the number of probes per unit area of the print surface is the print density. The print surface is that area of the substrate on which the individual probes are printed, plus the surface area between the individual probes. If there are two or more groupings of a substantial number of probes on surface of the substrate separated by surface area in which few or no probes are printed, the print surface includes the surface area between probes of a group but not the surface area of the substrate between groupings. Preferably, the print density is high so that a large number of probes can fit on a substrate. Preferably, the print density is at least about 200, 500, 1,000, 5,000, 10,000, 20,000, or 40,000 probes per cm2.
The probes of the probe microarray may be oligonucleotides (the term xe2x80x9coligonucleotidesxe2x80x9d as used herein also includes polynucleotides, especially polynucleotides having more than about 40 bases), or the probes may be proteins, cells, or chemical compounds. A microarray may contain any number of probes, and preferably the number of probes in the microarray is at least about 1,000, 5,000, 10,000, 50,000, 100,000, or 500,000. A probe microarray may be formed by attaching any of the probes discussed above individually to beads, which beads are affixed to the substrate: covalently; non-covalently through e.g. ionic, polar, or Van der Waals forces or conformational interaction of binding moieties attached to the beads and substrate (such as biotin-avidin or biotin-streptavidin); magnetically; or any other method for attaching beads to a substrate.
One method of the invention forms a probe microarray on a substrate. This method comprises the acts of: providing a print head having a bundle of individual capillaries; passing non-identical probe-containing liquids through a number of the capillaries simultaneously; and printing the non-identical probe-containing liquids onto the substrate to form the probe microarray. The probe-containing liquids may contain the probes in a suitable liquid carrier, or the probe-containing liquids may contain probes attached to e.g. beads such as magnetic beads that are deposited onto the substrate using a magnetic field to move the beads through the capillaries.
The individual capillaries of the bundle may be light-conducting capillaries. For instance, a light-conducting capillary is formed of a transparent material and has a properly designed refractive index profile across its cross section so that the capillary transports light from the distal end to the proximal end of the capillary. The capillary can therefore conduct light and fluid individually or simultaneously.
In one embodiment of the invention, a light-conducting capillary has a first portion having a first refractive index and a second portion having a second refractive index whose value is greater than the first refractive index wherein said second portion is inside the first portion. The light-conducting capillary further has a proximal end, a distal end, an axis, an inner wall defining a channel through the capillary, and an outer wall. The inner wall extends coaxially with the axis of the capillary, and the outer wall also extends coaxially with the axis of the capillary. The first portion and the second portion are configured such that a light beam launched into the proximal end is transmitted along the capillary and exits the capillary at the distal end. The channel of the capillary has a cross-sectional area that is sufficiently large that a fluid entering the channel at the proximal end of the capillary discharges at the distal end of the capillary. In one instance, a light-conducting capillary is formed by selecting a liquid carrier which has a refractive index that is sufficiently high compared to the refractive index of the capillary that the liquid acts as a light-conductive core and the capillary acts as cladding. Preferably, a light-conducting capillary is an optical fiber capillary, in which the capillary itself is configured to be light-conducting by providing a region of high refractive index along the length of the capillary that is bounded by regions of lower refractive index. The optical fiber capillary may be formed of doped silica, for instance. The cross-sectional area and outer diameter of the capillary is such that at least about 1000, 10,000, 100,000, or 500,000 non-overlapping spots of liquid may be deposited in an area of 12 cm2 on a substrate by bundling capillaries together. A bundle of light-conducting capillaries may be formed, and the bundle may be utilized as part of a print-head or printing system as described herein.
A capillary as used in a print head of the invention typically has a large ratio of length to outer diameter. The length of a capillary can be at least about 20 cm, and preferably at least about 100 cm. A capillary as used in the invention typically has an outer diameter less than 200 micron and preferably less than 100 micron. Consequently, the ratio of length to outer diameter ranges can be the ratio of any of these values, and typically the ratio of length to outer diameter is greater than 500, 4000, 10,000, or 30,000.
Thus, this invention features a unique carrier that simultaneously conduct light and transport minute quantity of material. The light can be used to carry information and/or energy. Individual carriers may be used as medical devices (e.g., for observing and treating diseased tissues or organs) or industrial devices (e.g., for inspecting and treating cracks or leaks). A plurality of a carrier can be bundled together to provide massive parallel capability in handling multiple samples and multiple information channels.
Light may be conducted through light-conducting capillaries of a print head before depositing probes or during probe deposition to e.g. prepare a light-sensitive area to receive the probes. Light may be conducted through the light-conducting capillaries of a print head during probe deposition to measure the distance between the capillary facet and the substrate and to detect in real time whether the probe fluid contacts the substrate surface. Light may be conducted through the capillaries after depositing probes as a quality control measure to determine if probes have been deposited, especially where some of the molecules of each probe incorporate a tag that fluoresces when illuminated with light of the appropriate wavelength. Preferably, the facet of the print head used to print the random probe microarray has at least about 83, 416, 833, 4166, 8333, or 41,666 capillaries per square centimeter. An electric potential may optionally be applied across the capillaries to move the probes in the probe-containing liquids through the capillaries. A probe microarray of the invention can be formed using any of the methods specified above.
A probe microarray of this invention may also comprise a substrate that is coated with a layer of light sensitive material, and a plurality of probes (i.e. spots of probe molecules) on a surface of the substrate. A light sensitive material may be hydrophobic but turn hydrophilic upon exposure to light of the appropriate wavelength. Probes can be more easily positioned on a portion of the substrate that is hydrophilic if the liquid in which probe molecules are carried is polar (e.g. water).
The invention also provides a method of using the probe microarrays discussed herein. The method includes contacting a probe microarray with a liquid which contains target components for a sufficient period of time to allow target components in the liquid to associate with complementary probes of the probe microarray, if any, to form target-probe complexes, and determining the positions of the target-probe complexes in the microarray. The positions may be correlated with a probe identity or with a target identity using, e.g., a software file or dedicated memory such as read-only memory that contains data on the probe and/or target identities as a function of probe position on the substrate.
In addition, the invention provides systems and methods of printing microarrays, even when the substrate and print head are not perfectly aligned and would otherwise not print a complete microarray of probes that the print head is capable of printing.
The invention further provides quality control instruments and methods for inspecting microarrays after their formation.
In one method of detecting the unintentional absence of probes from a probe microarray or the unintentional overlapping of adjacent probes, or mis-sizing of probe spots on the array, the method comprises positioning a microarray beneath a light detector and shining light on a probe-containing surface of the microarray at an angle to the microarray. The angle is sufficient to reflect light from the probe-containing surface in a first area of the surface that contains no probes. The angle is also sufficient to scatter light to the detector in a second area of the surface that contains probes. A light pattern array formed by scattering the light to the detector is detected, and the light pattern array is compared to an expected pattern array to determine if the light pattern array matches the expected pattern array.
In another method of detecting the unintentional absence of probes from a probe microarray or the unintentional overlapping of adjacent probes, or the mis-sizing of probe spots on the array, the method comprises positioning a microarray beneath a light detector and shining light on a surface of the microarray at an angle sufficient to cause total internal reflection of the light within the microarray. A light pattern array is formed by detecting the light refracting from within the microarray at a probe-containing area of the microarray and comparing the light pattern array to an expected pattern array to determine if the light pattern array matches the expected pattern array.
The invention also provides quality control instruments. One instrument detects the unintentional absence of probes from a probe microarray or the unintentional overlapping of adjacent probes, or the mis-sizing of probe spots on the array. This quality control instrument has a light detector and a light source configured to shine light onto a probe-containing surface of the microarray at a first angle to the microarray. The light contacting a first set of areas of the probe-containing surface that contain no probes reflects away from the light detector. The light contacting a second set of probe-containing areas of the probe-containing surface is scattered sufficiently that the detector detects the presence of the light at the second set of areas. A microprocessor receives data signals from the light detector, which data signals correspond to a light pattern array formed by the light scattered from said probe-containing areas of the microarray. The microprocessor is configured to compare the data signals corresponding to the light pattern array to data corresponding to an expected pattern array to determine if the light pattern array matches the expected pattern array.
Another quality control instrument of the invention also detects the unintentional absence of probes from a probe microarray or the unintentional overlapping of adjacent probes, or the mis-sizing of probe spots on the array. This quality control instrument has a light detector and a light source configured to shine light onto a surface of a microarray placed beneath the light detector. The light shines at an angle sufficient to cause total internal reflection of the light within the microarray. A microprocessor receives data signals from the light detector, which data signals correspond to a light pattern array formed by the light refracting from within the microarray at probe-containing areas of the microarray. The microprocessor is configured to compare the data signals corresponding to the light pattern array to data corresponding to an expected pattern array to determine if the light pattern array matches the expected pattern array.
A preferred arrayer based on the invention is simple and low cost and capable of producing one high-density (down to 10 xcexcm probe pitch), large scale (500,000 or more probes per slide) microarray in a single stamping action. The production throughput for a single arrayer can be as high as 5, 10 or 20 slides per second. Such a throughput gives it advantage in production of high volume and standard microarray products. In addition, it has great flexibility for custom microarrays as the entire or part of the capillaries in the stamp can be quickly washed clean and reused for different probe samples.
The invention thus provides a number of systems, components, means, and methods for producing probe microarrays as are more fully described below. This Summary section of the disclosure provides a summary of some salient points of the invention, but this section is not to be interpreted as limiting the scope of the invention to only those features and embodiments discussed in this section. Instead, the invention involves all components, systems, and methods discussed in this and the following sections in addition to those defined by the appended claims.